We performed global expression profiling of miRNAs before and after the differentiation of primary murine white and brown preadipocytes into mature adipocytes, in order to discover post-transcriptional regulators of adipogenesis. We found 65 miRNAs differentially expressed following differentiation of preadipocytes into adipocytes, but only subtle differences in fold-change of miRNAs between white and brown adipocytes, suggesting that the identified miRNAs most likely play a role in adipogenesis per se rather than in cell lineage determination. Ontological analysis of the target genes of the miRNAs regulated during in vitro adipogenesis indicated that processes related to transcription and metabolism are potentially targeted by the alteration in miRNA expression. One of the identified adipogenesis-regulated miRNAs, mir-21, showed a positive correlation with obesity in subcutaneous adipose tissue from healthy humans and this merits further investigation to verify the location of mir-21 expression in situ, in adipose tissue.
MiRNAs regulated by adipogenesis
Fiftyone miRNAs were significantly upregulated and fourteen downregulated during the differentiation of both white and brown preadipocytes. The trend for differences in the degree of miRNA upregulation or downregulation between the white and brown adipocytes was 2-fold. MiR-143 and miR-181a were expressed in both white and brown adipocytes during both developmental stages and showed a trend for a 2-fold enrichment in white adipocytes, similarly to what was found by Walden et al. . However, from this data set, differences in miRNA regulation between white and brown adipocyte population were very modest. 'Muscle-enriched' miRNAs, miR-1 and miR-133a, previously shown by Walden et al. to be modestly yet expressed in brown versus white adipocytes (but not upregulated during their differentiation), were too low in expression to be detected by this microarray study.
Two of the six miRNAs showed a trend for a stronger upregulation during white adipocyte differentiation - miR-24-1* and miR-23b. They belong to a recently identified miR-23b cluster shown to be a molecular switch regulating
response of liver stem cells during their differentiation , and also to be regulated by bone morphogenetic protein-2 (BMP-2) in mesenchymal stem cells differentiating into adipocytes . The BMP-2 signalling pathway plays a role in white adipocyte lineage determination, as BMP-2 can induce commitment of C3H10T1/2 pluripotent stem cells into white adipocytes . Our results, in combination with these previous observations, suggest that this mechanism might therefore involve miR-24-1 and miR-23b. Another important member of this miR-23b cluster is miR-27b, which has also been shown to play important roles in adipogenesis by impairing differentiation of human adipocytes and targeting PPARγ .
However, we found that miR-27b was not robustly expressed in brown or white primary adipocytes. Two miRNA, miR-669c and miR-34c, demonstrated a trend for downregulation during the maturation of white but not brown adipocytes. miR-669c has recently been implicated in ageing  and correlates with the decline of liver regeneration. The only miRNA that was significantly downregulated in both brown and white adipocytes (yet more so during the brown adipocyte maturation) was let-7e, which belongs to the let-7 family. Let-7 impairs adipogenic differentiation , explaining its downregulation during the adipogenic process. Consistent with in vitro adipogenesis, which consists of two processes - cell proliferation followed by differentiation - most of the miRNAs regulated by adipogenesis in our system are involved in the regulation of these two processes.
Evaluation of the similarity to other models through literature study
Ortega et al. examined global miRNA expression profiles during human adipogenesis and whether its pattern significantly differed between cells from obese or lean subjects . The differentiation protocol they used differed significantly from ours. They cultured human preadipocytes and passed them through 3 passages before the differentiation was induced at confluency using a cocktail of human Insulin, Dexamethasone, Isobutylmethyl-xanthine (IBMX), and the PPARγ agonist Rosiglitazone, in the presence of fetal bovine serum. This cocktail of drugs is usually necessary to promote successful differentiation in clonal cell lines. The cells we isolated from fat tissues were plated and the differentiation was induced spontaneously in media supplemented with only insulin and newborn bovine serum, and as such it perhaps provides a more native differentiation context. In Ortega's findings most of the miRNAs showed increased or decreased regulation after 7 days of differentiation. They identified the cluster of miRNAs related to miR-30 (miR-30a, b, c, d and e) as being increased during human white adipocyte maturation and these data are in accordance with our observations . Ortega et al., however, show miR-503 as the most down-regulated miRNA during differentiation of human adipocytes , while in our murine data set miR-503 was upregulated during both primary brown and white adipocyte differentiation.. The discrepancy with respect to mir-503 regulation may relate to differential regulation between man and mouse, or differences in the differentiation protocols applied.
Our results are also mostly in agreement with those of Esau et al.  who identified a similar expression pattern regarding miR-130b, miR-30c, miR-30a*, miR-191, miR-30d, miR-196, miR-30b, miR-19b, miR-92, miR-138 and miR-100 during differentiation of cultured human adipocytes. However, our results are not in accordance with Esau regarding miR-20, miR-93, miR-103 and miR-107. Esau et al., similarly to Ortega et al., used a cocktail of drugs to induce the differentiation of human preadipocytes. Thus, these discrepancies could again reflect differences between human and mouse adipogenesis or differences in in vitro culture conditions. Xie et al.  identified miR-143, miR-148a, miR-30c, miR146b as being upregulated during in vitro differentiation of 3T3-L1 cells, which is identical to our findings. However, Xie et al. additionally found miR-107 and miR-103 to be upregulated, similarly to Ortega's findings in human adipocytes, but contrary to our results. As the regulation of mir-107 and mir-103 exists in man and mouse, differences in the differentiation protocols applied could explain the discrepancy. One must also consider the time when such miRNA analysis was run and the possibility of alterations in the annotation of both miRNA sequence and chip design.
One example of miRNAs that can be actively involved in cell proliferation is the miR-17-92 cluster, which comprises seven miRNAs (miR-17-5p, miR-17-3p, miR-18, miR-19a, miR-20, miR-19b, and miR-92-1). This miR-17-92 cluster is frequently amplified in B-cell lymphomas and lung cancers, and promotes tumour growth in human and mouse cell models [45, 46]. Wang et al. used 3T3-L1 preadipocyte cells to screen miRNA expression over seven timepoints after hormonal induction . They found all five members of the miR-17-92 cluster to be markedly upregulated after hormonal stimulation, peaking at the clonal expansion stage. In our dataset, similarly, all members of the cluster were upregulated, suggesting that this cluster might play a role during both proliferation and differentiation of adipocytes.
Predicted protein targets and network analysis
To better understand the biological consequences of simultaneous changes in multiple miRNAs we utilised a weighted score ranking analysis method , which identifies the protein targets most likely to be regulated by collective changes in miRNA expression. We found the ontologies, transcription regulation and regulation of cellular metabolic processes, were strongly represented in the miRNA target list. We made a provisional assessment of the connectivity of the genes belonging to the ontology category 'SP_PIR_Transcription Regulation' using the literature co-citation network PubGene. Sirt1 was linked directly to 5 other genes from the analysed group. Sirt1, an NAD+-dependent deacetylase, belongs to the family of sirtuins, and regulates key aspects of lipid metabolism. Sirt1 plays a role in 3T3-L1 adipogenesis, as Sirt1 protein levels increase during adipogenesis, and overexpression of Sirt1 prevents adipogenesis by inhibiting PPARγ transactivation of adipogenesis-related genes, while knock-down of Sirt1 promotes fat accumulation . Activation of Sirt1 increases the release of FFA in both mature 3T3-L1 adipocytes and in primary white rat adipocytes . Sirt1 also appears to exert an anti-inflammatory effect and improves insulin sensitivity in 3T3-L1 adipocytes by promoting insulin-stimulated glucose uptake . Accordingly, Sirt1 activators improve insulin resistance in ob/ob and diet-induced obese mice, and increase insulin sensitivity in obese, insulin-resistant Zucker rats . Sirt1 genetic variation in humans is related to BMI and risk of obesity . The miRNAs targeting Sirt1 include miR-143, miR-23b miR-34c as well as mir-34a [51, 52].
Adipogenesis-regulated miRNAs and human obesity
Of the miRNAs we described in the murine primary cell cultures, miR-21 and miR-143 were differentially expressed in healthy non-obese persons (BMI <30) versus obese persons (BMI >30). Furthermore, we found a robust positive correlation of mir-21 with BMI in healthy humans. In the ob/ob mouse, which develops obesity and type 2 diabetes-like symptoms, expression of mir-21 in liver is downregulated  suggesting that mir-21 may have different roles depending on the cell type. Both miR-21 and miR-143 show an altered expression level in cancer: miR-21 is upregulated 5-fold in colorectal cancer, when compared with adjacent non-tumour tissue, while miR-143 is downregulated 3-fold . In human hepatocellular cancer , and in breast tumours , miR-21 is also overexpressed, when compared with normal tissue. An anti-miR-21 strategy decreases tumour growth in vivo, possibly by enhancing apoptosis . Cell proliferation is increased by miR-21 overexpression, while it is decreased by an anti-miR-21 strategy, when modest changes in miR-21 expression are achieved (2 to 4-fold changes) . In obese subjects there is an expansion of fat tissue, and while mir-21 is positively correlated to BMI, it is unknown whether mir-21 can induce adipose tissue expansion, or if it is increased as a consequence of adipose tissue expansion or the inclusion of additional cell types within the tissue. Gabriely et al. found that downregulation of mir-21 in glioma cells reduces their invasive potential, probably by relieving mir-21 targeting of metalloprotease inhibitors, allowing metallo proteases to be active . These data thus suggest that mir-21 could be important for tissue expansion, albeit this will require substantial experimental verification.